Immunostimulatory oligonucleotide multimers

ABSTRACT

The invention provides an immunostimulatory nucleic acid. In certain embodiments according to this aspect of the invention, the sequence of the immunostimulatory oligonucleotide and/or immunomer is at least partially self-complementary.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/892,550, filed Jul. 15, 2004 and a continuation of U.S.patent application Ser. No. 11/153,054, filed Jun. 15, 2005, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/579,985,filed Jun. 15, 2004, and U.S. Provisional Application Ser. No.60/599,362, filed on Aug. 6, 2004. The entire teachings of theabove-referenced Applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to immune stimulation by oligonucleotide analogs.

2. Summary of the Related Art

Tokunaga et al., J. Natl. Cancer Inst. 72 (1984) 955-96; Pisetsky etal.; Reich et al., Mol. Biol. Rep. 18 (1993) 217-221; Krieg et al., Yiet al., Nature 374 (1995) 546-549 and Sato et al., Science 273 (1996)352-354 teach that bacterial DNA, synthetic oligodeoxynucleotides, andDNA vaccines containing unmethylated CpG-dinucleotides in specificsequence contexts (CpG DNA) activate the vertebrate immune system.

Krieg et al., Annu. Rev. Immunol. 20 (2002) 709-760; Dalpke et al.,Biol. Chem. 383 (2002) 1491-1500 and Kandimalla et al., Curr. Opin. Mol.Ther. 4 (2002) 122-129 teach that CpG DNAs induce innate immune cells toproduce Th1 cytokines that promote cytotoxic T lymphocyte (CTL)responses and production of immunoglobulins by B cells. Theimmunostimulatory properties of CpG DNAs have allowed their use astherapeutic agents for a broad spectrum of disease indications includingcancers, viral and bacterial infections, inflammatory disorders and asadjuvant in immunotherapy.

In addition to chemical modifications, a number of structuralmodifications influenced the activity of CpG DNAs. Kandimalla et al.,Nucleic Acids Res. 30 (2002) 4460-4469 teaches that CpG DNAs thatcontained two freely accessible 5′-ends through a 3′-3′-linkage hadgreater activity than did conventional CpG DNAs containing multiplecopies of CpG motifs and a single 5′-end.

Kandimalla et al, Biochem. Biophys. Res. Commun. 306 (2003) 948-953teaches that the presence of a secondary structure in CpG DNAssignificantly affected their activity depending on the position andnature of the secondary structure, that the presence of a hairpinstructure at the 5′-end abrogated stimulatory activity, and that thesame structure at the 3′-end had an insignificant effect on stimulatoryactivity but caused lower IL-6 secretion and contributed to higherstability against nucleases.

There remains a need to “customize” the immune response throughmodification of oligonucleotide analogs.

BRIEF SUMMARY OF THE INVENTION

In a first aspect the invention provides an immunostimulatoryoligonucleotide the sequence of which is at least partiallyself-complementary. The immunostimulatory nucleic acid comprises anoligonucleotide sequence containing at least one dinucleotide selectedfrom the group consisting of CpG, C*pG, C*pG* and CpG*, wherein C iscytidine or 2′-deoxycytidine, G is guanosine or 2′-deoxyguanosine, C* is2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-dideoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substituted arabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate.

In some embodiments, the immunostimulatory nucleic acid is from about 2to about 50 nucleotides in length. In certain embodiments theimmunostimulatory nucleic acid is from about 12 to about 26 nucleotidesin length. In some embodiments, the oligonucleotides each have fromabout 3 to about 35 nucleoside residues, in further embodiments fromabout 4 to about 30 nucleoside residues, in even further embodimentsfrom about 4 to about 20 nucleoside residues. In some embodiments, theoligonucleotides have from about 5 to about 18, or from about 5 to about14, nucleoside residues. As used herein, the term “about” implies thatthe exact number is not critical. Thus, the number of nucleosideresidues in the oligonucleotides is not critical, and oligonucleotideshaving one or two fewer nucleoside residues, or from one to severaladditional nucleoside residues are contemplated as equivalents of eachof the embodiments described above. In some embodiments, one or more ofthe oligonucleotides have 11 nucleotides.

In a second aspect the invention provides an immunomer comprising atleast two oligonucleotides linked by a non-nucleotide linker, whereinthe sequences of the immunostimulatory oligonucleotides are at leastpartially self-complementary. In certain embodiments according to thisaspect of the invention at least one of the oligonucleotides contains atleast one dinucleotide selected from the group consisting of CpG, C*pG,C*pG* and CpG*, wherein C is cytidine or 2′-deoxycytidine, G isguanosine or 2′-deoxyguanosine, C* is 2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-dideoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substituted arabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate.

In some embodiments, the immunostimulatory nucleic acid is from about 2to about 50 nucleotides in length. In certain embodiments theimmunostimulatory nucleic acid is from about 12 to about 26 nucleotidesin length. In some embodiments, the oligonucleotides each have fromabout 3 to about 35 nucleoside residues, or from about 4 to about 30nucleoside residues, or from about 4 to about 20 nucleoside residues. Insome embodiments, the oligonucleotides have from about 5 to about 18, orfrom about 5 to about 14, nucleoside residues. As used herein, the term“about” implies that the exact number is not critical. Thus, the numberof nucleoside residues in the oligonucleotides is not critical, andoligonucleotides having one or two fewer nucleoside residues, or fromone to several additional nucleoside residues are contemplated asequivalents of each of the embodiments described above. In someembodiments, one or more of the oligonucleotides have 11 nucleotides.

In a third aspect the invention provides pharmaceutical compositions.These compositions comprise any one of the compositions disclosed in thefirst and second aspects of the invention either alone or in combinationand a pharmaceutically acceptable carrier.

In a fourth aspect the invention provides a method for generating animmune response in a vertebrate. This method comprises administering tothe vertebrate any one of the compositions, alone or in combination,disclosed in the first, second and third aspects of the invention. Thecompositions disclosed herein can be administered through any suitableroute of administration including, but not limited to, parenteral, oral,sublingual, transdermal, topical, intranasal, aerosol, intraocular,intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye dropand mouthwash.

In a fifth aspect the invention provides a method for therapeuticallytreating a vertebrate having cancer, an autoimmune disorder, airwayinflammation, inflammatory disorders, skin disorders, allergy, asthma ora disease caused by a pathogen. This method comprises administering tothe vertebrate any one of the compositions, alone or in combination,disclosed in the first, second and third aspects of the invention. Thecompositions disclosed herein can be administered through any suitableroute of administration including, but not limited to, parenteral, oral,sublingual, transdermal, topical, intranasal, aerosol, intraocular,intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop,ear drop and mouthwash.

In a sixth aspect the invention provides a method for preventing cancer,an autoimmune disorder, airway inflammation, inflammatory disorders,skin disorders, allergy, asthma or a disease caused by a pathogen in avertebrate. This method comprises administering to the vertebrate anyone of the compositions, alone or in combination, disclosed in thefirst, second and third aspects of the invention. The compositionsdisclosed herein can be administered through any suitable route ofadministration including, but not limited to, parenteral, oral,sublingual, transdermal, topical, intranasal, aerosol, intraocular,intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop,ear drop and mouthwash.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are representations of various embodiments of theinvention (SEQ ID NO: 41). In FIG. 1B, m and n are independently 0-1000.

FIG. 2 is a synthetic scheme for the parallel synthesis of immunomers ofthe invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 3 depicts a group of representative small molecule linkers suitablefor linear synthesis of immumomers of the invention.

FIG. 4 is a synthetic scheme for the linear synthesis of immunomers ofthe invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 5 depicts a group of representative small molecule linkers suitablefor parallel synthesis of immunomers of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The issued patents, patent applications, and references that are citedherein are hereby incorporated by reference to the same extent as ifeach was specifically and individually indicated to be incorporated byreference. In the event of inconsistencies between any teaching of anyreference cited herein and the present specification, the latter shallprevail for purposes of the invention.

The invention relates to the therapeutic use of oligonucleotides asimmunostimulatory agents for immunotherapy applications. The inventionalso provides methods for generating, enhancing and modifying the immuneresponse caused by immunostimulatory compounds used for immunotherapyapplications such as, but not limited to, treatment and/or prevention ofcancer, autoimmune disorders, asthma, respiratory allergies, foodallergies, and bacteria, parasitic, and viral infections in adult andpediatric human and veterinary applications. Allergic asthma is acertain embodied condition for treatment by the present methods andcompounds. Thus, the invention further provides compounds having optimallevels of immunostimulatory effect for immunotherapy and methods formaking and using such compounds. In addition, immunostimulatoryoligonucleotides/immunomers of the invention are useful as adjuvants incombination with DNA vaccines, antibodies, allergens, chemotherapeuticagents, and antisense oligonucleotides.

In a first aspect the invention provides an immunostimulatoryoligonucleotide the sequence of which is at least partiallyself-complementary. The immunostimulatory nucleic acid comprises annucleic acid sequence containing at least one dinucleotide selected fromthe group consisting of CpG, C*pG, C*pG* and CpG*, wherein C is cytidineor 2′-deoxycytidine, G is guanosine or 2′-deoxyguanosine, C* is2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-dideoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substituted arabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate.

In some embodiments, the immunostimulatory oligonucleotide is from about2 to about 50 nucleotides in length. In certain embodiments theimmunostimulatory oligonucleotide is from about 12 to about 26nucleotides in length. In some embodiments, the oligonucleotides arefrom about 3 to about 35 nucleoside residues, or from about 4 to about30 nucleoside residues, or from about 4 to about 20 nucleoside residues.In some embodiments, the oligonucleotides have from about 5 to about 18,or from about 5 to about 14, nucleoside residues. As used herein, theterm “about” implies that the exact number is not critical. Thus, thenumber of nucleoside residues in the oligonucleotides is not critical,and oligonucleotides having one or two fewer nucleoside residues, orfrom one to several additional nucleoside residues are contemplated asequivalents of each of the embodiments described above. In someembodiments, one or more of the oligonucleotides have 11 nucleotides.

As would be recognized by one skilled in the art, the complementarysequence of the oligonucleotides allows for intermolecular hydrogenbonding thereby giving the oligonucleotides secondary structure.Additional oligonucleotides can bind together thereby creating a chain,or multimers, of oligonucleotides according to the invention.

In a second aspect the invention provides an immunomer comprising atleast two oligonucleotides linked by a non-nucleotide linker, whereinthe sequences of the immunostimulatory oligonucleotides are at leastpartially self-complementary. In certain embodiments according to thisaspect of the invention at least one of the oligonucleotides contains atleast one dinucleotide selected from the group consisting of CpG, C*pG,C*pG* and CpG*, wherein C is cytidine or 2′-deoxycytidine, G isguanosine or 2′-deoxyguanosine, C* is 2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-dideoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substituted arabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate.

In this aspect, immunostimulatory nucleic acid comprises a structure asdetailed in formula (I).Domain A-Domain B-Domain C  (I)

Domains may be from about 2 to about 12 nucleotides in length. Domain Amay be 5′-3′ or 3′-5′ or 2′-5′ DNA, RNA, RNA-DNA, DNA-RNA having apalindromic or self-complementary domain containing or not containing atleast one dinucleotide selected from the group consisting of CpG, C*pG,C*pG* and CpG*, wherein C is cytidine or 2′-deoxycytidine, G isguanosine or 2′-deoxyguanosine, C* is 2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-deoxy-5-halocytosine, 2′-deoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate. In certain embodiments, the immunostimulatorydinucleotide is not CpG.

In certain embodiments, Domain A will have more than one dinucleotideselected from the group consisting of CpG, C*pG, C*pG* and CpG*.

Domain B, as depicted by an “X” below, is a linker joining Domains A andC that may be a 3′-'5′ linkage, a 2′-5′ linkage, a 3′-3′ linkage, aphosphate group, a nucleoside, or a non-nucleoside linker that may bealiphatic, aromatic, aryl, cyclic, chiral, achiral, a peptide, acarbohydrate, a lipid, a fatty acid, mono- tri- or hexapolyethyleneglycol, or a heterocyclic moiety.

Domain C may be 5′-3′ or 3′-5′, 2′-5′ DNA, RNA, RNA-DNA, DNA-RNA PolyI-Poly C having a palindromic or self-complementary sequence, containingor not containing a dinucleotide selected from the group consisting ofCpG, C*pG, C*pG*, CpG*, wherein C is cytidine or 2′-deoxycytidine, G isguanosine or 2′-deoxyguanosine, C* is 2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-deoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substituted arabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate. In certain embodiments, the immunostimulatorydinucleotide is not CpG. In some embodiments, Domain B is preferably anon-nucleotidic linker connecting oligonucleotides of Domain A andDomain C, which are referred to as “immunomers.” In certain embodiments,Domain C does not have the dinucleotide CpG, C*pG, C*pG* or CpG*.

In some embodiments, the oligonucleotides contained in formula (I) arefrom about 2 to about 50 nucleotides in length. In certain embodimentsthe oligonucleotides contained in formula (I) are from about 12 to about26 nucleotides in length. In some embodiments, the oligonucleotides eachhave from about 3 to about 35 nucleoside residues, preferably from about4 to about 30 nucleoside residues, more preferably from about 4 to about20 nucleoside residues. In some embodiments, the oligonucleotides havefrom about 5 to about 18, or from about 5 to about 14, nucleosideresidues. As used herein, the term “about” implies that the exact numberis not critical. Thus, the number of nucleoside residues in theoligonucleotides is not critical, and oligonucleotides having one or twofewer nucleoside residues, or from one to several additional nucleosideresidues are contemplated as equivalents of each of the embodimentsdescribed above. In some embodiments, one or more of theoligonucleotides have 11 nucleotides.

A self-complementary sequence as used herein refers to a base sequencewhich, upon suitable alignment, may form intramolecular or, moretypically, intermolecular basepairing between G-C, A-T, A-U and/or G-Uwobble pairs. In one embodiment the extent of self-complementarity is atleast 50 percent. For example an 8-mer that is at least 50 percentself-complementary may have a sequence capable of forming 4, 5, 6, 7, or8 G-C, A-T, A-U and/or G-U wobble basepairs. Such basepairs may but neednot necessarily involve bases located at either end of theself-complementary immunostimulatory oligonucleotide and/or immunomer.Where nucleic acid stabilization may be important to theimmunostimulatory oligonucleotide and/or immunomer, it may beadvantageous to “clamp” together one or both ends of a double-strandednucleic acid, either by basepairing or by any other suitable means. Thedegree of self-complementarity may depend on the alignment betweenimmunostimulatory oligonucleotide and/or immunomer, and such alignmentmay or may not include single- or multiple-nucleoside overhangs. Inother embodiments, the degree of self-complementarity is at least 60percent, at least 70 percent, at least 80 percent, at least 90 percent,or even 100 percent.

Similar considerations apply to intermolecular basepairing betweenimmunostimulatory oligonucleotides and/or immunomers of different basesequence. Thus, where a plurality of immunostimulatory oligonucleotidesand/or immunomers are used together, the plurality of immunostimulatoryoligonucleotides and/or immunomers may, but need not, include sequenceswhich are at least partially complementary to one another. In oneembodiment the plurality of immunostimulatory oligonucleotides and/orimmunomers includes an immunostimulatory oligonucleotide and/orimmunomer having a first sequence and an immunostimulatoryoligonucleotide and/or immunomer having a second sequence, wherein thefirst sequence and the second sequence are at least 50 percentcomplementary. For example, as between two 8-mers that are at least 50percent complementary, they may form 4, 5, 6, 7, or 8 C-C, A-T, A-U,and/or G-U wobble basepairs. Such basepairs may but need not necessarilyinvolve bases located at either end of the complementaryimmunostimulatory oligonucleotides and/or immunomers. The degree ofcomplementarity may depend on the alignment between immunostimulatoryoligonucleotides and/or immunomers, and such alignment may or may notinclude single- or multiple-nucleoside overhangs. In other embodiments,the degree of complementarity is at least 60 percent, at least 70percent, at least 80 percent, at least 90 percent, or even 100 percent.

By way of non-limiting example, in certain embodiments of this aspectthe immunostimulatory nucleic acid will have a structure as detailed informula (II).

As would be recognized by one skilled in the art, the depictedimmunostimulatory nucleic acid/immunomer compounds have secondarystructure because the sequences of the domains are complementaryallowing for intermolecular hydrogen bonding. Domains A and A′ may ormay not be identical, domains A and C may or may not be identical,domains A and C′ may or may not be identical, domains A′ and C may ormay not be identical, domains A′ and C′ may or may not be identical,domains B and B′ may or may not be identical and domains C and C′ may ormay not be identical. Moreover, as shown in FIG. 1, additionalimmunomers can bind through intermolecular hydrogen bonding therebycreating a chain, or multimers, of immunomers according to theinvention. n can be any number of continuous, self complementaryimmunomer compounds.

As used herein, the term “complementary” means having the ability tohybridize to a nucleic acid. Such hybridization is ordinarily the resultof hydrogen bonding between complementary strands, preferably to formWatson-Crick or Hoogsteen base pairs, although other modes of hydrogenbonding, as well as base stacking can also lead to hybridization.

As used herein, the term “secondary structure” refers to intermolecularhydrogen bonding. Intermolecular hydrogen bonding results in theformation of a duplexed nucleic acid molecule.

“Palindromic sequence” shall mean an inverted repeat (i.e., a sequencesuch as ABCDEE′D′C′B′A′ in which A and A′, B and B′, etc., are basescapable of forming the usual Watson-Crick base pairs. In vivo, suchsequences may form double-stranded structures. In one embodiment the CpGnucleic acid contains a palindromic sequence. A palindromic sequenceused in this context refers to a palindrome in which the CpG is part ofthe palindrome. In some embodiments the CpG is the center of thepalindrome. In another embodiment the CpG nucleic acid is free of apalindrome. An immunostimulatory nucleic acid that is free of apalindrome is one in which the CpG dinucleotide is not part of apalindrome. Such an oligonucleotide may include a palindrome in whichthe CpG is not the center of the palindrome.

For purposes of the invention, the term “oligonucleotide” refers to apolynucleoside formed from a plurality of linked nucleoside units. Sucholigonucleotides can be obtained from existing nucleic acid sources,including genomic or cDNA, but are preferably produced by syntheticmethods. In some embodiments each nucleoside unit includes aheterocyclic base and a pentofuranosyl, 2′-deoxypentfuranosyl,trehalose, arabinose, 2′-deoxy-2′-substituted arabinose,2′-O-substituted arabinose or hexose sugar group. The nucleosideresidues can be coupled to each other by any of the numerous knowninternucleoside linkages. Such internucleoside linkages include, withoutlimitation, phosphodiester, phosphorothioate, phosphorodithioate,alkylphosphonate, alkylphosphonothioate, phosphotriester,phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate,carbamate, morpholino, borano, thioether, bridged phosphoramidate,bridged methylene phosphonate, bridged phosphorothioate, and sulfoneinternucleoside linkages. The term “oligonucleotide” also encompassespolynucleosides having one or more stereospecific internucleosidelinkage (e.g., (RP)- or (SP)-phosphorothioate, alkylphosphonate, orphosphotriester linkages). As used herein, the terms “oligonucleotide”and “dinucleotide” are expressly intended to include polynucleosides anddinucleosides having any such internucleoside linkage, whether or notthe linkage comprises a phosphate group. In certain embodiments, theseinternucleoside linkages may be phosphodiester, phosphorothioate, orphosphorodithioate linkages, or combinations thereof.

The term “oligonucleotide” also encompasses polynucleosides havingadditional substituents including, without limitation, protein groups,lipophilic groups, intercalating agents, diamines, folic acid,cholesterol and adamantane. The term “oligonucleotide” also encompassesany other nucleobase containing polymer, including, without limitation,peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholino-backboneoligonucleotides, and oligonucleotides having backbone sections withalkyl linkers or amino linkers.

The oligonucleotides of the invention can include naturally occurringnucleosides, modified nucleosides, or mixtures thereof. As used herein,the term “modified nucleoside” is a nucleoside that includes a modifiedheterocyclic base, a modified sugar moiety, or a combination thereof. Insome embodiments, the modified nucleoside is a non-natural pyrimidine orpurine nucleoside, as herein described. In some embodiments, themodified nucleoside is a 2′-substituted ribonucleoside anarabinonucleoside or a 2′-deoxy-2′-substituted-arabinoside.

For purposes of the invention, the term “2′-substituted ribonucleoside”or “2′-substituted arabinoside” includes ribonucleosides orarabinonucleosides in which the hydroxyl group at the 2′ position of thepentose moiety is substituted to produce a 2′-substituted or2′-O-substituted ribonucleoside. In certain embodiments, suchsubstitution is with a lower alkyl group containing 1-6 saturated orunsaturated carbon atoms, or with an aryl group having 6-10 carbonatoms, wherein such alkyl, or aryl group may be unsubstituted or may besubstituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro,acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examplesof 2′-O-substituted ribonucleosides or 2′-O-substituted-arabinosidesinclude, without limitation 2′-O-methylribonucleosides or2′-O-methylarabinosides and 2′-O-methoxyethoxyribonucleosides or2′-O-methoxyethoxyarabinosides.

The term “2′-substituted ribonucleoside” or “2′-substituted arabinoside”also includes ribonucleosides or arabinonucleosides in which the2′-hydroxyl group is replaced with a lower alkyl group containing 1-6saturated or unsaturated carbon atoms, or with an amino or halo group.Examples of such 2′-substituted ribonucleosides or 2′-substitutedarabinosides include, without limitation, 2′-amino, 2′-fluoro, 2′-allyl,and 2′-propargyl ribonucleosides or arabinosides.

The term “oligonucleotide” includes hybrid and chimericoligonucleotides. A “chimeric oligonucleotide” is an oligonucleotidehaving more than one type of internucleoside linkage. One non-limitingexample of such a chimeric oligonucleotide is a chimeric oligonucleotidecomprising a phosphorothioate, phosphodiester or phosphorodithioateregion and non-ionic linkages such as alkylphosphonate oralkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. Nos.5,635,377 and 5,366,878).

A “hybrid oligonucleotide” is an oligonucleotide having more than onetype of nucleoside. One non-limiting example of such a hybridoligonucleotide comprises a ribonucleotide or 2′ substitutedribonucleotide region, and a deoxyribonucleotide region (see, e.g.,Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

Alternatively, the nucleic acid molecule of the invention can be twoimmunomers linked by way of a non-nucleotidic linker.

In certain embodiments of the invention, at least one immunostimulatoryoligonucleotide of the invention comprises an immunostimulatorydinucleotide of the formula 5′-Pyr-Pur-3′, wherein Pyr is a naturalpyrimidine nucleoside or analog thereof and Pur is a natural purinenucleoside or analog thereof. As used herein, the term “pyrimidinenucleoside” refers to a nucleoside wherein the base component of thenucleoside is a pyrimidine base. Similarly, the term “purine nucleoside”refers to a nucleoside wherein the base component of the nucleoside is apurine base. For purposes of the invention, a “synthetic” pyrimidine orpurine nucleoside includes a non-naturally occurring pyrimidine orpurine base, a non-naturally occurring sugar moiety, or a combinationthereof.

In certain embodiments pyrimidine nucleosides in the immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention have the structure (III):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, hydrogen bond acceptor, hydrophilic group, hydrophobic group,electron withdrawing group and electron donating group;

A is a hydrogen bond acceptor or a hydrophilic group;

A′ is selected from the group consisting of hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group;

X is carbon or nitrogen; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

In certain embodiments, the sugar ring is derivatized with a phosphatemoiety, modified phosphate moiety, or other linker moiety suitable forlinking the pyrimidine nucleoside to another nucleoside or nucleosideanalog.

In some embodiments hydrogen bond donors include, without limitation,—NH—, —NH₂, —SH and —OH. Preferred hydrogen bond acceptors include,without limitation, C═O, C═S, and the ring nitrogen atoms of an aromaticheterocycle, e.g., N3 of cytosine.

In some embodiments, the base moiety in (III) is a non-naturallyoccurring pyrimidine base. Examples of preferred non-naturally occurringpyrimidine bases include, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, or N4-ethylcytosine, and4-thiouracil. In some embodiments, the sugar moiety S′ in (III) is anon-naturally occurring sugar moiety. For purposes of the presentinvention, a “naturally occurring sugar moiety” is a sugar moiety thatoccurs naturally as part of nucleic acid, e.g., ribose and2′-deoxyribose and a “non-naturally occurring sugar moiety” is any sugarthat does not occur naturally as part of a nucleic acid, but which canbe used in the backbone for an oligonucleotide, e.g, hexose. Arabinoseand arabinose derivatives are non-limiting examples of sugar moieties.

In some embodiments purine nucleoside analogs in immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention have the structure (IV):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, and hydrophilic group;

A is a hydrogen bond acceptor or a hydrophilic group;

X is carbon or nitrogen;

each L is independently selected from the group consisting of C, O, Nand S; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

In certain embodiments, the sugar ring is derivatized with a phosphatemoiety, modified phosphate moiety, or other linker moiety suitable forlinking the pyrimidine nucleoside to another nucleoside or nucleosideanalog.

In certain embodiments hydrogen bond donors include, without limitation,—NH—, —NH₂, —SH and —OH. Preferred hydrogen bond acceptors include,without limitation, C═O, C═S, —NO₂ and the ring nitrogen atoms of anaromatic heterocycle, e.g., N1 of guanine.

In some embodiments, the base moiety in (IV) is a non-naturallyoccurring purine base. Examples of preferred non-naturally occurringpurine bases include, without limitation, 6-thioguanine and7-deazaguanine. In some embodiments, the sugar moiety S′ in (IV) is anaturally occurring sugar moiety, as described above for structure(III).

In a third aspect the invention provides pharmaceutical compositions.These compositions comprise any one of the compositions disclosed in thefirst and second of the invention either alone or in combination and apharmaceutically acceptable carrier.

As used herein, the term “physiologically acceptable” refers to amaterial that does not interfere with the effectiveness of thecompositions of the first, second or third aspects of the invention andis compatible with a biological system such as a cell, cell culture,tissue, or organism. In certain embodiments, the biological system is aliving organism, such as a vertebrate.

As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, or other materialwell known in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication. The preparation of pharmaceutically acceptable formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990, ISBN: 0-912734-04-3.

Pharmaceutical compositions of the invention may also include a cancervaccine, including a cancer vaccine selected from EFG, Anti-idiotypiccancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV gangliosideconjugate vaccine, Her2/new, Ovarex® (oregovomab), M-Vax, O-Vax, L-Vax,STn-KHL Theratope® (STn-KLH), BLP25 (MUC-1), liposomal idiotypicvaccine, Melacine® (melanoma theraccine), peptide antigen vaccines,toxin/antigen vaccines, MVA-vased vaccine, PACIS, BCG vaccine, TA-HPV,TA-CIN, DISC-virus and ImmunCyst/TheraCys® (BCG Live (Intravesical)).

In various embodiments of the invention, the compositions of the first,second or third aspects of the invention may be covalently linked to anantigen or otherwise operatively associated with an antigen. As usedherein, the term “operatively associated with” refers to any associationthat maintains the activity of both the compositions of the first,second or third aspects of the invention and the antigen. Non-limitingexamples of such operative associations include being part of the sameliposome or other such delivery vehicle or reagent. In embodimentswherein the compositions of the first, second or third aspects of theinvention are covalently linked to an antigen, such covalent linkage isat any position on the compositions of the first, second or thirdaspects of the invention other than an accessible 5′ end of animmunostimulatory oligonucleotide. For example, the antigen may beattached at an internucleoside linkage or may be attached to thenon-nucleotidic linker. Alternatively, the antigen may itself be thenon-nucleotidic linker.

In various embodiments of the invention, the compositions of the first,second or third aspects of the invention may include an oligonucleotidewith antisense activity. As used herein, “antisense activity” means thatthe oligonucleotide, when introduced into a cell or an animal, causes areduction in the expression of the gene to which it is complementary.

In various embodiments of the invention, the compositions of the first,second or third aspects of the invention may include an oligonucleotidesequence that is an aptamer. Aptamers are nucleic acid molecules thathave been selected from random pools based on their ability to bindother molecules. Aptamers have been selected which bind nucleic acids,proteins, small organic compounds, and even entire organisms. Thesenovel molecules have many potential uses in medicine and technology(see, e.g., Burgstaller P., et al. Curr Opin Drug Discov Devel. 5:690-700 (2002)).

The pharmaceutical compositions of the invention may be administered byany suitable route, including, without limitation, parenteral, oral,sublingual, transdermal, topical, intranasal, aerosol, intraocular,intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eyedrop or mouthwash form. The pharmaceutical compositions can be deliveredusing known procedures at dosages and for periods of time effectiveobtain the desired effect, e.g. the treatment of cancer, the treatmentof infection and the treatment of autoimmune-diseases. When administeredsystemically, the pharmaceutical compositions are administered at asufficient dosage to attain a blood level of the compositions of thefirst, second and/or third aspects of the invention from about 0.0001micromolar to about 10 micromolar. For localized administration, muchlower concentrations than this may be effective, and much higherconcentrations may be tolerated. In certain embodiments, a total dosageof immunostimulatory oligonucleotide and/or immunomer ranges from about0.0001 mg per patient per day to about 200 mg per kg body weight perday. It may be desirable to administer simultaneously, or sequentially atherapeutically effective amount of one or more of the therapeuticcompositions of the invention to an individual as a single treatmentepisode.

Immunostimulatory oligonucleotides were created as immunomers using thefollowing protocols for synthesis. The immunostimulatoryoligonucleotides and/or immunomers of the invention may conveniently besynthesized using an automated synthesizer and phosphoramidite approachas schematically depicted in FIGS. 2 and 4. In some embodiments, theimmunostimulatory oligonucleotides and/or immunomers are synthesized bya linear synthesis approach (see FIG. 2). Representative linkers forthis synthesis are presented in FIG. 3. As used herein, the term “linearsynthesis” refers to a synthesis that starts at one end of the immunomerand progresses linearly to the other end. Linear synthesis permitsincorporation of either identical or un-identical (in terms of length,base composition and/or chemical modifications incorporated) monomericunits into the immunostimulatory oligonucleotides and/or immunomers.

An alternative mode of synthesis for immunostimulatory oligonucleotidesand/or immunomers is “parallel synthesis”, in which synthesis proceedsoutward from a central linker moiety (see FIG. 4). Representativelinkers for this method of synthesis are presented in FIG. 5. A solidsupport attached linker can be used for parallel synthesis, as isdescribed in U.S. Pat. No. 5,912,332. Alternatively, a universal solidsupport, such as phosphate attached to controlled pore glass support,can be used.

Parallel synthesis of immunostimulatory oligonucleotides and/orimmunomers has several advantages over linear synthesis: (1) parallelsynthesis permits the incorporation of identical monomeric units; (2)unlike in linear synthesis, both (or all) the monomeric units aresynthesized at the same time, thereby the number of synthetic steps andthe time required for the synthesis is the same as that of a monomericunit; and (3) the reduction in synthetic steps improves purity and yieldof the final immunomer product.

At the end of the synthesis by either linear synthesis or parallelsynthesis protocols, the immunostimulatory oligonucleotides orimmunomers according to the invention may conveniently be deprotectedwith concentrated ammonia solution or as recommended by thephosphoramidite supplier, if a modified nucleoside is incorporated. Theproduct immunostimulatory oligonucleotides and/or immunomer ispreferably purified by reversed phase HPLC, detritylated, desalted anddialyzed.

The compositions disclosed in the first second and third aspects of theinvention can comprise the immunostimulatory oligonucleotide orimmunomer alone or as oligonucleotide/immunomer conjugates. Anoligonucleotide/immunomer conjugate comprises an oligonucleotide orimmunomer, as described above, and an antigen conjugated to theoligonucleotide and/or immunomer at a position other than the accessible5′ end. In some embodiments, the non-nucleotidic linker comprises anantigen, which is conjugated to the oligonucleotide. In some otherembodiments, the antigen is conjugated to the oligonucleotide at aposition other than its 3′ end. In some embodiments, the antigenproduces a vaccine effect. The immunostimulatory oligonucleotide orimmunomer alone or as oligonucleotide/immunomer conjugates can beadministered in the methods discussed below.

The antigen is optionally selected from antigens associated with apathogen, antigens associated with a cancer, antigens associated with anauto-immune disorder, and antigens associated with other diseases suchas, but not limited to, veterinary or pediatric diseases, or wherein theantigen is an allergen. For purposes of the invention, the term“associated with” means that the antigen is present when the pathogen,cancer, auto-immune disorder, food allergy, skin allergy, respiratoryallergy, asthma or other disease is present, but either is not present,or is present in reduced amounts, when the pathogen, cancer, auto-immunedisorder, food allergy, skin allergy, respiratory allergy, or disease isabsent.

The immunomer is covalently linked to the antigen, or it is otherwiseoperatively associated with the antigen. As used herein, the term“operatively associated with” refers to any association that maintainsthe activity of both immunomer and antigen. Nonlimiting examples of suchoperative associations include being part of the same liposome or othersuch delivery vehicle or reagent. In embodiments wherein the immunomeris covalently linked to the antigen, such covalent linkage preferably isat any position on the immunomer other than an accessible 5′ end of animmunostimulatory oligonucleotide. For example, the antigen may beattached at an internucleoside linkage or may be attached to thenon-nucleotidic linker. Alternatively, the antigen may itself be thenon-nucleotidic linker.

In a fourth aspect, the invention provides methods for generating and/ormodulating an immune response in a vertebrate, such methods comprisingadministering to the vertebrate an immunomer or immunomer conjugateaccording to the invention. In some embodiments, the vertebrate is amammal. For purposes of this invention, the term “mammal” is expresslyintended to include humans. In certain embodiments, the immunomer orimmunomer conjugate is administered to a vertebrate in need ofimmunostimulation.

As used herein, the term “modulating” or “modulate” means to increase ordecrease the immunostimulatory activity of an immunostimulatory nucleicacid relative to that of the parent immunostimulatory nucleic acid.

In the methods according to this aspect of the invention, administrationof immunomers can be by any suitable route, including, withoutlimitation, parenteral, oral, sublingual, transdermal, topical,intranasal, intramuscular, intraperitonal, subcutaneous, intradermal,aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun,dermal patch or in eye drop or mouthwash form. Administration of thetherapeutic compositions of immunomers can be carried out using knownprocedures at dosages and for periods of time effective to reducesymptoms or surrogate markers of the disease. When administeredsystemically, the therapeutic composition is preferably administered ata sufficient dosage to attain a blood level of immunomer from about0.0001 micromolar to about 10 micromolar. For localized administration,much lower concentrations than this may be effective, and much higherconcentrations may be tolerated. Preferably, a total dosage of immunomerranges from about 0.001 mg per patient per day to about 200 mg per kgbody weight per day. It may be desirable to administer simultaneously,or sequentially a therapeutically effective amount of one or more of thetherapeutic compositions of the invention to an individual as a singletreatment episode.

Either the immunomer or the vaccine, or both, may optionally be linkedto an immunogenic protein, such as keyhole limpet hemocyanin (KLH),cholera toxin B subunit, or any other immunogenic carrier protein ornonimmunogenic carrier protein. Any of the plethora of adjuvants may beused including, without limitation, Freund's complete adjuvant, Freund'sincomplete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, andsaponins, including QS-21, imiquimod, R848, or combinations thereof.

Toll-like receptors (TLRs) function as sensors of infection and inducethe activation of innate and adaptive immune responses. TLRs recognize awide variety of ligands, called pathogen-associated molecular patterns(PAMPs). Upon recognizing conserved pathogen-associated molecularproducts, TLRs activate host defense responses through theirintracellular signaling domain, the Toll/interleukin-1 receptor (TIR)domain, and the downstream adaptor protein MyD88. Dendritic cells andmacrophages normally respond to Toll-like receptor (TLR) ligands andcytokines (for example, interleukin-1β; IL-6 and tumor necrosis factor,TNF), which they also produce; natural killer (NK) cells and T cells arealso involved in the pro-inflammatory circuit. After TLR stimulation bybacterial compounds, innate immune cells release a range of cytokines.Some examples of TLR ligands include, but are not limited to,lipoproteins; peptidoglycan, zymosan (TLR2), double-stranded RNA,polyI:polyC (TLR3), lipopolysaccharide, heat shock proteins, Taxol®(paclitaxel) (TLR4), flagellin (TLR5), and imidazoquinolines-R848,resiquimod, imiquimod; ssRNA (TLR7/8), beta-lymphocytes (TLR10) anduropathogenic E. coli (TLR11).

The methods according to this aspect of the invention are useful formodel studies of the immune system. The methods are also useful for theprophylactic or therapeutic treatment of human or animal disease. Forexample, the methods are useful for pediatric and veterinary vaccineapplications.

In a fifth aspect, the invention provides methods for therapeuticallytreating a vertebrate having a disease or disorder, such methodscomprising administering to the vertebrate an immunomer or immunomerconjugate according to the invention. In various embodiments, thedisease or disorder to be treated is cancer, an autoimmune disorder,airway inflammation, inflammatory disorders, allergy, asthma or adisease caused by a pathogen. Pathogens include bacteria, parasites,fungi, viruses, viroids and prions. Administration is carried out asdescribed for the fourth aspect of the invention.

For purposes of the invention, the term “allergy” includes, withoutlimitation, food allergies atopic dermatitis, allergic rhinitis (alsoknown as hay fever), allergic conjunctivitis, urticaria (also known ashives), respiratory allergies and allergic reactions to other substancessuch as latex, medications and insect stings or problems commonlyresulting from allergic rhinitis-sinusitis, otitis media and COPD. Theterm “airway inflammation” includes, without limitation, asthma.Specific examples of asthma include, but are not limited to, allergicasthma, non-allergic asthma, exercised-induced asthma, occupationalasthma, and nocturnal asthma.

Allergic asthma is characterized by airway obstruction associated withallergies and triggered by substances called allergens. Triggers ofallergic asthma include, but are not limited to, airborne pollens,molds, animal dander, house dust mites and cockroach droppings.Non-allergic asthma is caused by viral infections, certain medicationsor irritants found in the air, which aggravate the nose and airways.Triggers of non-allergic asthma include, but are not limited to,airborne particles (e.g., coal, chalk dust), air pollutants (e.g.,tobacco smoke, wood smoke), strong odors or sprays (e.g., perfumes,household cleaners, cooking fumes, paints or varnishes), viralinfections (e.g., colds, viral pneumonia, sinusitis, nasal polyps),aspirin-sensitivity, and gastroesophageal reflux disease (GERD).Exercise-induced asthma (EIA) is triggered by vigorous physicalactivity. Symptoms of EIA occur to varying degrees in a majority ofasthma sufferers and are likely to be triggered as a result of breathingcold, dry air while exercising. Triggers of EIA include, but are notlimited to, breathing airborne pollens during exercise, breathing airpollutants during exercise, exercising with viral respiratory tractinfections and exercising in cold, dry air. Occupational asthma isdirectly related to inhaling irritants and other potentially harmfulsubstances found in the workplace. Triggers of occupational asthmainclude, but are not limited to, fumes, chemicals, gases, resins,metals, dusts, vapors and insecticides.

As used herein, the term “autoimmune disorder” refers to disorders inwhich “self” proteins undergo attack by the immune system. Such termincludes autoimmune asthma.

Without wishing to be bound to any particular theory, decreased exposureto bacteria may be partially responsible for the increased incidence of,severity of, and mortality due to allergic diseases such as asthma,atopic dermatitis, and rhinitis in the developed countries. Thishypothesis is supported by evidence that bacterial infections orproducts can inhibit the development of allergic disorders inexperimental animal models and clinical studies. Bacterial DNA orsynthetic oligodeoxynucleotides containing unmethylated CpGdinucleotides and/or modified CpG dinucleotides in certain sequencecontexts (CpG DNA) potently stimulate innate immune responses andthereby acquired immunity. The immune response to CpG DNA includesactivation of innate immune cells, proliferation of B cells, inductionof Th1 cytokine secretion, and production of immunoglobulins (Ig). Theactivation of immune cells by CpG DNA occurs via Toll-like receptor 9(TLR9), a molecular pattern recognition receptor. CpG DNA induce strongTh1-dominant immune responses characterized by secretion of IL-12 andIFN-γ. Immunomers (IMO) alone or as allergen conjugates decreaseproduction of IL-4, IL-5, and IgE and reduce eosinophilia in mousemodels of allergic asthma. IMO compounds also effectively reverseestablished atopic eosinophilic airway disease by converting a Th2response to a Th1 response.

OVA with alum is commonly used to establish a Th2-dominant immuneresponse in various mouse and rat models. The Th2 immune responseincludes increased IL-4, IL-5, and IL-13 production, elevated serumlevels of total and antigen-specific IgE, IgG1, and lower levels ofIgG2a. IMO compounds prevent and reverse established Th2-dominant immuneresponses in mice. The co-administration of IMO compounds with OVA/alumto mice reduces IL-4, IL-5, and IL-13 production and induces IFN-γproduction in spleen-cell cultures subjected to antigen re-stimulation.Furthermore, IMO compounds inhibit antigen-specific and total IgE andenhance IgG2a production in these mice.

Injection of OVA/alum and IMO compounds induces a lymphocyteantigen-recall response (Th1-type) in mice characterized by low levelsof Th2-associated cytokines, IgE and IgG1, and high levels ofTh1-associated cytokines and IgG2a. Co-administration of IMO compoundswith other kinds of antigens, such as S. masoni egg and hen egglysozyme, also result in reversal of the Th2-response to a Th1-dominantresponse in in vitro and in vivo studies. As described herein, IMOcompounds effectively prevent development of a Th2 immune response andallow a strong Th1 response.

While Th2 cytokines trigger an Ig isotype switch towards production ofIgE and IgG1, the Th1 cytokine IFN-γ induces production of IgG2a byB-lymphocytes. Mice injected with OVA/alum and IMO compounds producelower levels of IL-4, IL-5, and IL-13 and higher levels of IFN-γ,accompanied by lower IgE and IgG1 and higher IgG2a levels, than miceinjected with OVA/alum alone. This suggests the existence of a closelink between Th1-cytokine induction and immunoglobulin isotype switch inmice that receive antigen and IMO compounds.

Serum antigen-specific and total IgE levels are significantly lower inmice receiving OVA/alum and IMO compounds than in mice receivingOVA/alum alone. In contrast, OVA-specific IgG1 levels areinsignificantly changed and total IgG1 levels are only slightlydecreased compared with mice injected with OVA/alum alone (data notshown). The different response may result from different mechanismsinvolved in the control of IgE and IgG1 class switch, though bothisotypes are influenced by IL-4 and IL-13. For example, IL-6 promotes Blymphocytes to synthesize IgG1 in the presence of IL-4.

In a sixth aspect the invention provides a method for preventing cancer,an autoimmune disorder, airway inflammation, inflammatory disorders,skin disorders, allergy, asthma or a disease caused by a pathogen in avertebrate. This method comprises administering to the vertebrate anyone of the compositions, alone or in combination, disclosed in thefirst, second and third aspects of the invention. Pathogens includebacteria, parasites, fungi, viruses, viroids and prions. Administrationis carried out as described for the fourth aspect of the invention.

In any of the methods according to the invention, the immunostimulatoryoligonucleotide and/or immunomer or a conjugate thereof can beadministered in combination with any other agent useful for treating thedisease or condition that does not diminish the immunostimulatory effectof the oligonucleotide or immunomer. For purposes of this aspect of theinvention, the term “in combination with” means in the course oftreating the same disease in the same patient, and includesadministering the oligonucleotide and/or immunomer and an agent in anyorder, including simultaneous administration, as well as any temporallyspaced order, for example, from sequentially with one immediatelyfollowing the other to up to several days apart. Such combinationtreatment may also include more than a single administration of theimmunomer, and independently the agent. The administration of theoligonucleotide and/or immunomer and agent may be by the same ordifferent routes.

In any of the methods according to the invention, the agent useful fortreating the disease or condition includes, but is not limited to,vaccines, antigens, antibodies, cytotoxic agents, allergens,antibiotics, antisense oligonucleotides, peptides, proteins, genetherapy vectors, DNA vaccines and/or adjuvants to enhance thespecificity or magnitude of the immune response, or co-stimulatorymolecules such as cytokines, chemokines, protein ligands,trans-activating factors, peptides and peptides comprising modifiedamino acids. Additionally, the agent can include DNA vectors encodingfor antigen or allergen. In these embodiments, the immunomers of theinvention can variously act as adjuvants and/or produce directimmunostimulatory effects.

The examples below are intended to further illustrate certain preferredembodiments of the invention, and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1 Oligonucleotide Synthesis, Purification and ThermalMelt Profiles

CpG oligonucleotides (immunostimulatory oligonucleotides/immunomers)were synthesized on a 1 to 2 μmole scale usingβ-cyanoethylphosphoramidites on a PerSeptive Biosystem's 8909 ExpediteDNA synthesizer (PerSeptive Biosystem, Boston, Mass.). Thephosphoramidites of dA, dG, dC, and T were obtained from PE Biosystems(Foster City, Calif.). As described by lyer R. P., et al. (J. Am. Chem.Soc. 112: 1253-1254 (1990)), an iodine oxidizing agent was used toobtain the phosphorothioate backbone modification. All oligos weredeprotected using standard protocols, purified by HPLC, and dialyzedagainst USP quality sterile water for irrigation. The oligos werelyophilized and dissolved again in distilled water and theconcentrations were determined from UV absorbance at 260 nm. All oligoswere characterized by CGE and MALDI-TOF mass spectrometry (AppliedBiosystem's Voyager-DETM STR Biospectrometry™ Workstation) for purityand molecular mass, respectively. The purity of full-length oligosranged from 90-96% with the rest being shorter by one or two nucleotides(n-1 and n-2) as determined by CGE and/or denaturing PAGE. All oligoscontained less than <0.1 EU/mL of endotoxin as determined by the Limulusassay (Bio-Whittaker now known as Cambrex Bio Science Walkersville,Inc., Walkersville, Md.).

Thermal melting studies were carried out in 1 mL solution of 10 mMdisodium hydrogen phosphate, pH 7.2±0.2, containing 150 mM NaCl, and 2mM MgCl2. The solutions were heated to 95° C. for 10 min and allowed tocome to room temperature slowly before being stored at 4° C. overnight.The final concentration of oligonucleotide strand was 2.0 μM. UV thermalmelting measurements were performed at 260 nm on a Perkin-Elmer Lambda20 Spectrophotometer attached to a peltier thermal controller and apersonal computer using 1 cm path length quartz cuvettes at a heatingrate of 0.5° C./min. Melting temperatures (Tm) were taken as thetemperature of half-dissociation and were obtained from first derivativeplots. Each Tm value is an average of two or three independentexperiments and the values were within ±1.0° C.

Example 2 Cell Culture Conditions and Reagents

Spleen cells from 4-8 week old BALB/c, C57BL/6 or C3H/HeJ mice werecultured in RPMI complete medium as described by Zhao, Q., et al.(Biochem Pharmacol. 51: 173-182 (1996)) and Branda, R. F., et al.(Biochem. Pharmacol. 45: 2037-2043 (1993)). Murine J774 macrophages(American Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209) were cultured in Dulbecco's modified Eagles mediumsupplemented with 10% (v/v) fetal calf serum and antibiotics (100 IU/mLof penicillin G/streptomycin). All other culture reagents were purchasedfrom Mediatech (Gaithersburg, Md.).

Example 3 Spleen Cell Proliferation Assay

Typically, mouse (Balb-C) spleen cells were cultured with immunomercompounds at concentrations of 0.1, 1.0, and 10.0 μg/ml for 48 h andcell proliferation was determined by 3H-uridine incorporation, asdescribed by Zhao, Q., et al. (Biochem Pharmacol. 51: 173-182 (1996)).

Example 4 Cytokine Induction Assays

Mouse spleen or J774 cells were plated in 24-well dishes using 5×10⁶ or1×10⁶ cells/mL, respectively. The immunomer compounds dissolved in TEbuffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were added to a finalconcentration of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 μg/mL to the cellcultures. The cells were then incubated at 37° C. for 24 hr and thesupernatants were collected for ELISA assays. The experiments wereperformed two or three times for each immunomer compound and intriplicate for each concentration. The secretion of IL-12 and IL-6 wasmeasured by sandwich ELISA as described by Bhagat L., et al. (Biochem.Biophys. Res. Commun. 300: 853-861 (2003)). The required reagents,including cytokine antibodies and standards were purchased from BDBiosciences Pharmingen (San Diego, Calif.).

Example 5 Mouse Splenomegaly Assay

Female BALB/c mice (4-6 weeks, 19-21 gm) were divided into groups ofthree mice. Immunomer compounds were dissolved in sterile phosphatebuffered saline (PBS) and administered subcutaneously (SC) to mice at adose of 5 mg/kg. The mice were sacrificed after 48 hr and the spleenswere harvested and weighed as described by Zhao, Q., et al. (BiochemPharmacol. 51: 173-182 (1996)) and Branda, R. F., et al. (Biochem.Pharmacol. 45: 2037-2043 (1993)).

Example 6 Activation of the NF-κB Pathway

Toll-like receptor 9 (TLR9) has been shown to recognize unmethylatedCpG-dinucleotides in bacterial, plasmid and synthetic DNAs (Hemmi H., etal. Nature 408: 740-745 (2000)) and activate stress kinase (Yi A. K., etal. J. Immunol. 161: 4493-4497 (1998)) and NF-κB pathways (Stacey K. J.,et al. J. Immunol. 157: 2116-2122 (1996)). NF-κB activation in J774cells treated with immunomer compounds was carried out and analyzed byEMSA as described Yu D., et al. (Biochem. Biophys. Res. Commun. 297:83-90 (2002)) and Bhagat L., et al. (Biochem. Biophys. Res. Commun. 300:853-861 (2003)).

Example 7 Isolation of Human B Cells and Plasmacytoid Dendritic Cells(pDCs)

PBMCs from freshly drawn healthy volunteer blood (CBR Laboratories,Boston, Mass.) were isolated by Ficoll density gradient centrifugationmethod (Histopaque-1077, Sigma) and B cells were isolated from PBMCs bypositive selection using the CD19 cell isolation kit (Miltenyi Biotec)according to the manufacturer's instructions. Table 1 shows theimmunostimulatory activity of immunomer compounds of the invention inC57BL/6 Splenocyte Assay.

TABLE 1 Immunomer Structure and C57BL/6 Splenocyte Assay (24 hs) IL-12IL-12 IL-12 SEQ ID (pg/ml) (pg/ml) (pg/ml) NO Sequences and Modification(5′ 3′) 1 μg/ml 1 μg/ml 1 μg/ml 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 6396±10 911±61 and 34 22 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₂CTGTCT-5′ 7275±77 749±80 and 35 23 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTGC ₁TGTCT-5′ 8034±14  918±136 and 36 24 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTCCACTCT-5′ 752±98 and 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 275′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG ₁T-5′  389±59 28 5′-CTGTCG₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 717±25 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG₂oCTGTC-5′ 849±29 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media   75±28 104±7  IL-6 IL-6 IL-12 SEQ ID (pg/ml) (pg/ml) (pg/ml) NOSequences and Modification (5′ 3′) 1 μg/ml 1 μg/ml 1 μg/ml 19 5′-TCG₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 2195±77 423±99and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 3278±2   840±243 and35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 7080±0  1553±670 and 36 245′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′  693±226 and 37 25 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 1329±53 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 18±3 295′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 1230±83  30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 325′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG₁C-Y-GACACG ₁TGTCT-5′ and 39 Media  102±25 12±2 Normal phase representsa phosphorothioate linkage; o represents a phosphodiester linkage. G ₁= 2′-deoxy-7-deazaguanosine G ₂ = Arabinoguanosine C ₁= -(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker Y = C ₃Linker

Example 8 Human pDC Cultures and IFN-α and IFN-β ELISA

pDCs were isolated from human PBMCs using a BDCA-4 cell isolation kit(Miltenyi Biotec) according to the manufacturer's instructions. pDC wereplated in 96-well plates using 1×10⁶ cells/mL, 200 μL/well). Theimmunomer compounds were added to a final concentration of 0.3, 1.0,3.0, or 10.0 μg/mL to the cell cultures and incubated at 37° C. for 24hr. Supernatants were then harvested and assayed for IFN-α and IFN-βusing ELISA kit (PBL). Tables 2A-2D show an average ±SD of IFN-α andIFN-β for immunomer compounds according to the invention at aconcentration of 10.0 μg/mL.

TABLE 2A Immunomer Structure and Immunostimulatory Activity in Human DCAssay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN1 DN2 DN3 15′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 26112±604  25825±416 96264±605  2 5′-TCG ₁AACG ₁TTCG-X-GCTTG ₁CAAG ₁CT-5′ 20340±106 12270±306  105804±688  3 5′-TCTCACCTTCT-X-TCTTCCACTCT-5′ 185±0  311±4  1649±262 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG₂TTCG-X-GCTTG ₂CAAG ₂CT-5′ media 177±0  177±0    0±0 IFN-α IFN-α IFN-α(pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml NO Sequences andModification (5′–3′) DN4 DN5 DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG₁CT-5′ 41718±1015 25011±5    19608±5    2 5′-TCG ₁AACG ₁TTCG-X-GCTTG₁CAAG ₁CT-5′ 49176±302  14014±1414 21988±1413 35′-TCTCACCTTCT-X-TCTTCCACTCT-5′  0±0 197±0  201±0  4 5′-TCG ₂AACG ₂TTCG₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG ₂TTCG-X-GCTTG ₂CAAG ₂CT-5′ media 0±0 201±0  196±0  Normal phase represents a phosphorothioate linkageG= 2′-deoxy-7-deazaguanosine G ₂ = Arabinoguanosine X = Glycerol linker

TABLE 2B Immunomer Structure and Immunostimulatory Activity in Human DCAssay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN1 DN2 DN3 65′-TCG ₁TCG ₁AACG ₁TTCG ₁AGATGAT-3′ 37116±1108 44624±321 58908±707 75′-TCG ₂TCG ₂AACG ₂TTCG ₂AGATGAT-3′ 6606±950 5022±334 15637±698 8 5′-TCG₃TCG ₃AACG ₃TTC ₃AGATGAT-3′ 1405±121 7750±618 46311±506 9 5′-TC ₁GTC₁GAAC ₁GTTC ₁GAGATGAT-3′ 611±33 231±4   ±0 10 5′-TC ₂GTC ₂GAAC ₂GTTC₂GAGATGAT-3′ 269±7 185±0 2574±64 11 5′-TC ₃GTC ₃GAAC ₃GTTC ₃GAGATGAT-3′191±0‘0 188±0   0±0 media 177±0  177±0   0±0 IFN-α IFN-α IFN-α (pg/ml)(pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequences andModification (5′–3′) DN4 DN5 DN6 6 5′-TCG ₁TCG ₁AACG ₁TTCG ₁AGATGAT-3′98178±375 68722±1358 31678±715 7 5′-TCG ₂TCG ₂AACG ₂TTCG ₂AGATGAT-3′40782±885 19180±735  8696±1122 8 5′-TCG ₃TCG ₃AACG ₃TTCG ₃AGATGAT-3′12446±894 42195±2665 582±78 9 5′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′10175±206 15966±1256  6857±1335 10 5′-TC ₂GTC ₂GAAC ₂GTTC ₂GAGATGAT-3′13028±911 1947±204 30±5 11 5′-TC ₃GTC ₃GAAC ₃GTTC ₃GAGATGAT-3′   0±0355±16 17±0 media   0±0 12±0 10±0 Normal phase represents aphosphorothioate linkage G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine G ₃ = 2′-deoxyinosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine C ₂= Arabinocytidine C ₃ = 2′-deoxy-5-hydroxycytidine

TABLE 2C Immunomer Structure and Immunostimulatory Activity in Human DCAssay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN1 DN2 DN3 15′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 67088±306  35055±659 62805±328  40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 12588±448 19986±1418 38002±1087 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′16090±179  16712±584  90560±1690 13 5′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG₁CT-5′ 9092±291 9286±615 60570±867  14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 770±158 208±28 5529±286 15 5′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′40353±542  33164±419  72730±954  4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG₂CT-5′ 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′media 160±7  259±20  0±0 IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN4DN5 DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 66980±217 6552±1   7992±24  40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 83115±134 12 5-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 61230±1120 13 5′-TCG ₁TTCG ₁AACG₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 34430±451  14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′2044±62  15 5′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 33716±872  45′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 2440±23  2403±4   16 5′-TCG₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 1316±0 1683±10  17 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ media 546±0   0±0  0±0 IFN-α IFN-α IFN-α(pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequencesand Modification (5′–3′) DN7 DN8 DN9 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG₁CAAG ₁CT-5′ 31227±1341 9777±10  10008±10   40 5′-TCG ₁AACG₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 135′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 145′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 6083±184 16 5′-TCG₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 2164±4   17 5′-TCG ₁AACG ₂TTCG ₁-X-G₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5 media 0±0  0±0  0±0 IFN-α IFN-α (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml ID NOSequences and Modification (5′–3′) DN8 DN9 1 5′-TCG ₁AACG ₁TTCG ₁-X-G₁CTTG ₁CAAG ₁CT-5′ 1370±54  650±38 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG₁CT-5′ 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG ₁AACG₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG₂CAAG ₂CT-5′ 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG₁CT-5 media  0±0  0±0 Normal phase represents a phosphorothioate linkageG ₁ = 2′-deoxy-7-deazaguanosine G ₂ = Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker

TABLE 2D Immunomer Structure and Immunostimulatory Activity in Human DCAssay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN1 DN2 DN3 195′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 3145±4   5808±28  22050±407 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 4710±31  5656±0   14157±10   21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ and 34 22 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC₁TGTCT-5′ and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5 and 37 25 5′-TG₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media 228±0  234±0  116±0  IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN4DN5 DN6 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 16100±542  205′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 23768±1371 21 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 5824±530 2090±81  and 34 22 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 9582±49  1623±108 and 35 23 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 6912±157 1577±168 and 36 24 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTCCACTCT-5′ 19570±467  2254±25  and 37 25 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 305′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 125±3  157±0  179±0 IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/mlID NO Sequences and Modification (5′–3′) DN7 DN8 DN9 19 5′-TCG ₁AACG₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 1049±13  15594±48  6024±135 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 2230±78 6118±3   3162±189 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′1535±130 6680±35  1558±45  and 36 24 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTCCACTCT-5′ 16053±3815 9502±57  6228±0   and 37 25 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 6632±184 3166±242 295′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 6864±394 1146±42  30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 325′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 157±0  82±0 94±2 IFN-α IFN-α IFN-α(pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequencesand Modification (5′–3′) DN10 DN11 DN12 19 5′-TCG ₁AACG ₂TTCG ₁-X-G₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 215′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 8154±597 31854±136  and 34 225′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 6413±876 14493±613  9642±129 and35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 2688±293 4486±94  and 3624 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 7214±18  10068±31   and 37 255′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ 11474±402  and 34 26 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTCCACTCT-5′ 375±23 and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 5508±41013956±355  6009±240 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 5599±14611824±720   9977±1379 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′11946±159  31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′  0±0 32 5′-TCG₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ 10032±9    and 38 33 5′-TG ₁CAACG ₁CTTG₁C-Y-GACACG ₁TGTCT-5′ 6420±139 and 39 Media 80±0 101±7   0±0 Normalphase represents a phosphorothioate linkage; o represents aphosphodiester linkage. G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker Y = C ₃Linker

Example 9 Cytokine Analysis

The secretion of IFN-α in vertebrate cells, preferably BALB/c mousespleen cells or human PBMC, was measured by sandwich ELISA. The requiredreagents including cytokine antibodies and cytokine standards werepurchased form PharMingen, San Diego, Calif. ELISA plates (Costar) wereincubated with appropriate antibodies at 5 μg/mL in PBSN buffer(PBS/0.05% sodium azide, pH 9.6) overnight at 4° C. and then blockedwith PBS/1% BSA at 37° C. for 30 minutes. Cell culture supernatants andcytokine standards were appropriately diluted with PBS/10% FBS, added tothe plates in triplicate, and incubated at 25° C. for 2 hours. Plateswere overlaid with 1 μg/mL appropriate biotinylated antibody andincubated at 25° C. for 1.5 hours. The plates were then washedextensively with PBS-T Buffer (PBS/0.05% Tween 20) and further incubatedat 25° C. for 1.5 hours after adding streptavidin conjugated peroxidase(Sigma, St. Louis, Mo.). The plates were developed with Sure Blue™(Kirkegaard and Perry) chromogenic reagent and the reaction wasterminated by adding Stop Solution (Kirkegaard and Perry). The colorchange was measured on a Ceres 900 HD1 Spectrophotometer (Bio-TekInstruments).

Human peripheral blood mononuclear cells (PBMCs) were isolated fromperipheral blood of healthy volunteers by Ficoll-Paque density gradientcentrifugation (Histopaque-1077, Sigma, St. Louis, Mo.). Briefly,heparinized blood was layered onto the Histopaque-1077 (equal volume) ina conical centrifuge and centrifuged at 400×g for 30 minutes at roomtemperature. The buffy coat, containing the mononuclear cells, wasremoved carefully and washed twice with isotonic phosphate bufferedsaline (PBS) by centrifugation at 250×g for 10 minutes. The resultingcell pellet was then resuspended in RPMI 1640 medium containingL-glutamine (MediaTech, Inc., Herndon, Va.) and supplemented with 10%heat inactivated FCS and penicillin-streptomycin (100 U/ml). Cells werecultured in 24 well plates for different time periods at 1×10⁶cells/ml/well in the presence or absence of oligonucleotides. At the endof the incubation period, supernatants were harvested and stored frozenat −70° C. until assayed for various cytokines including IFN-α(BioSource International) by sandwich ELISA. The results are shown inTable 3A-3D below.

In all instances, the levels of IFN-α in the cell culture supernatantswas calculated from the standard curve constructed under the sameexperimental conditions for IFN-α.

TABLE 3A Immunomer Structure and Immunostimulatory Activity in HumanPBMC Assay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10μg/ml 10 μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN4 DN5DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 8222±44  6114±1  3604±1  2 5′-TCG ₁AACG ₁TTCG-X-GCTTG ₁CAAG ₁CT-5′ 6700±7   6272±24 2822±24 3 5′-TCTCACCTTCT-X-TCTTCCACTCT-5′  0±0 80±0  80±0 4 5′-TCG ₂AACG₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG ₂TTCG-X-GCTTG ₂CAAG ₂CT-5′media  0±0 78±0  83±0 IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10μg/ml 10 μg/ml 10 μg/ml ID NO Sequences and Modification (5′–3′) DN4 DN5DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 7038±11  2921±32112514±351 2 5′-TCG ₁AACG ₁TTCG-X-GCTTG ₁CAAG ₁CT-5′ 7332±269 3647±70410872±613 3 5′-TCTCACCTTCT-X-TCTTCCACTCT-5′ 19±7  0±0   0±0 4 5′-TCG₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG ₂TTCG-X-GCTTG ₂CAAG₂CT-5′ media 33±0  0±0   0±0 Normal phase represents a phosphorothioatelinkage G ₁ = 2′-deoxy-7-deazaguanosine G ₂ = Arahinoguanosine X= Glycerol linker

TABLE 3B Immunomer Structure and Immunostimulatory Activity in HumanPBMC Assay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10μg/ml 10 μg/ml 10 μg/ml ID NO Sequence and Modification (5′–3′) DN1 DN2DN3 6 5′-TCG ₁TCG ₁AACG ₁TTCG ₁AGATGAT-3′ 3487±1015 268±3  3883±50   75′-TCG ₂TCG ₂AACG ₂TTCG ₂AGATGAT-3′ 9±0 30±0 0±0 8 5′-TCG ₃TCG ₃AACG₃TTCG ₃AGATGAT-3′ 126±1    0±0 0±0 9 5′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′0±0  0±0 0±0 10 5′-TC ₂GTC ₂GAAC ₂GTTC ₂GAGATGAT-3′ 0±0 20±0 0±0 115′-TC ₃GTC ₃GAAC ₃GTTC ₃GAGATGAT-3′ 11±1   5±0 76±0  media 33±0   0±00±0 IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10μg/ml ID NO Sequence and Modification (5′–3′) DN4 DN5 DN6 6 5′-TCG ₁TCG₁AACG ₁TTCG ₁AGATGAT-3′ 1950±88   4342±225 426±85  7 5′-TCG ₂TCG ₂AACG₂TTCG ₂AGATGAT-3′ 10±0  1807±0   31±15 8 5′-TCG ₃TCG ₃AACG ₃TTCG₃AGATGAT-3′ 0±0 2876±344 48±5  9 5′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′ 0±0 5±0 4±0 10 5′-TC ₂GTC ₂GAAC ₂GTTC ₂GAGATGAT-3′ 0±0  8±0 5±3 11 5′-TC₃GTC ₃GAAC ₃GTTC ₃GAGATGAT-3′ 0±0 2111±330 11±3  media 0±0 48±9 11±2 Normal phase represents a phosphorothioate linkage G ₁= 2′-deoxy-7-deazaguanosine G ₂ = Arabinoguanosine G ₃ = 2′-deoxyinosineC ₁ = 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine C ₂= Arabinocytidine C ₃ = 2′-deoxy-5-hydroxycytidine

TABLE 3C Immunomer Structure and Immunostimulatory Activity in HumanPBMC Assay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10μg/ml 10 μg/ml 10 μg/ml ID NO Sequence and Modification (5′–3′) DN1 DN2DN3 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 208±33 432±5 1345±20 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 11±1 59±0 173±41 12 5′-TCG₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 16±1 55±7 324±49 13 5′-TCG ₁TTCG ₁AACG₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 882±32 733±80 2035±16  145′-TCCAACCTTCG-X-GCTTCCAACCT-5′  50±27  39±17  4±0 15 5′-TCG ₁TTG ₁CAACG₁-X-G ₁CAACG ₁TTG ₁CT-5′ 604±6  465±70 1902±30  4 5′-TCG ₂AACG ₂TTCG₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 175′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G₁CTTGC ₁AAG ₁CT-5 media 20±4 12±0  3±0 IFN-α IFN-α IFN-α (pg/ml) (pg/ml)(pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequence and Modification(5′–3′) DN4 DN5 DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 900±8 432±26 582±20 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 92±6 12 5′-TCG₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 441±76 13 5′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG₁CTTG ₁CT-5′ 732±8 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 19±5 15 5′-TCG₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 880±8  4 5′-TCG ₂AACG ₂TTCG ₂-X-G₂CTTG ₂CAAG ₂CT-5′ 27±0 26±0 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′19±0 23±0 17 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ media  6±1  0±0  0±0 IFN-α IFN-α IFN-α(pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NO Sequenceand Modification (5′–3′) DN7 DN8 DN9 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG₁CAAG ₁CT-5′ 324±18 578±28 741±25 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG₁CT-5′ 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG ₁AACG₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG₂CAAG ₂CT-5′  6±0 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′  0±0 175′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G₁CTTGC ₁AAG ₁CT-5′ media  0±0  0±0  0±0 Normal phase represents aphosphorothioate linkage G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker

TABLE 3D Immunomer Structure and Immunostimulatory Activity in HumanPBMC Assay (24 hs) IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10μg/ml 10 μg/ml 10 μg/ml ID NO Sequence and Modification (5′–3′) DN1 DN2DN3 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′  8±0 65±3 314±23 205′-TCG ₁AACGTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′  9±0 10±2 487±87 21 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG₂CTGTCT-5′ and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ and 36 245′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ and 37 25 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 305′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 11±0 10±0  0±0 IFN-αIFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NOSequence and Modification (5′–3′) DN4 DN5 DN6 19 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 1446±7    20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC₁AAG ₁CT-5′ 942±1  21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5 126±2 159±13 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 239±23  356±109and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 147±23 185±46 and 3624 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 107±15 148±37 and 37 25 5′-TG₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media  0±0 68±5 67±0 IFN-α IFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10μg/ml 10 μg/ml 10 μg/ml ID NO Sequence and Modification (5′–3′) DN7 DN8DN9 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG,-Y-TCTTG ₁CTGTCT-5′242±1  549±37  9±0 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′241±2  250±12 14±1 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′238±0  224±25  8±1 and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′238±0  668±10 41±7 and 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′223±3  112±0  29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 47±4  5±1 305′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39 Media  0±0  0±0  2±0 IFN-αIFN-α IFN-α (pg/ml) (pg/ml) (pg/ml) SEQ 10 μg/ml 10 μg/ml 10 μg/ml ID NOSequence and Modification (5′–3′) DN10 DN11 DN12 19 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-521 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 232±8  252±16 and 34 225′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 67±1 195±3  364±8  and 35 235′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 70±1 148±3  and 36 24 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 443±29  678±133 and 37 25 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ 298±16 and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ 12±1 and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′  2±0 94±5 512±33 295′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′  3±0  61±18 168±25 30 5′-TCG₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 992±2  31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG₁TGTCG ₁T-5′  9±0 32 5′-TCG ₁AACG ₁TTCG,-Y-GACAG ₁CTGTCT-5′ 1528±20  and38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′  69±10 and 39 Media  0±0 6±0  7±0 Normal phase represents a phosphorothioate linkage; orepresents a phosphodiester linkage. G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker Y = C ₃Linker

Example 10 Flow Cytometric Analysis

Cell surface markers of CD69 and CD86 were detected with a CoulterEpics-XL Flow Cytometer using anti-human CD69-Fitc and CD86-Fitc, whichwere purchased from BD Pharmingen (San Diego, USA). Staining methodswere briefly described as follow. The activated culture cells wereblocked with 10% Human AB serum (Sigma) in staining buffer (PBS with 1%BSA and 0.1% NaN₃) at 4° C. for 1 hour and stained with the antibodiesat 4° C. overnight. PBMCs (4×10⁵) were stained with CD69-Fitc andCD86-Fitc. PDCs (2×10⁵) were stained CD86-Fitc. The cell staining datawere acquired and analyzed with Coulter System II software (see Tables4A-4F below).

TABLE 4A Immunomer Structure and Expression of BC from Human PBMC (2× 10⁶ cell/ml) (24 hs) %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml IDNO Sequences and Modification (5′–-3′) DN1 DN2 DN3 1 5′-TCG ₁AACG ₁TTCG₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 36.4 17.7 36.4 2 5′-TCG ₁AACG ₁TTCG-X-GCTTG₁CAAG ₁CT-5′2 7.2 6.3 30.4 3 5′-TCTCACCTTCT-X-TCTTCCACTCT-5′ 15 7.3 11.34 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG₂TTCG-X-GCTTG ₂CAAG ₂CT-5′ media 10.7 8 7.6 %CD86 %CD86 %CD86 SEQ 1μg/ml 1 μg/ml 1 μg/ml ID NO Sequences and Modification (5′–-3′) DN4 DN5DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 33.3 12.9 27.6 25′-TCG ₁AACG ₁TTCG-X-GCTTG ₁CAAG ₁CT-5′ 25 17.5 T 32.4 35′-TCTCACCTTCT-X-TCTTCCACTCT-5′ 10 21.3 117.6 4 5′-TCG ₂AACG ₂TTCG ₂-X-G₂CTTG ₂CAAG ₂CT-5′ 5 5-TCG ₂AACG ₂TTCG-X-GCTTG ₂CAAG ₂CT-5′ media 5.2 5411.8 %CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences andModification (5′–-3′) DN1 DN2 DN3 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG₁CT-5′ 27.5 61 75.8 2 5′-TCG ₁AACG ₁TTCG-X-GCTTG ₁CAAG ₁CT-5′ 52.5 46.569.7 3 5′-TCTCACCTTCT-X-TCTTCCACTCT-5′ 0 11.8 8.5 4 5′-TCG ₂AACG ₂TTCG₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG ₂TTCG-X-GCTTG ₂CAAG ₂CT-5′ media0 11.1 11.1 %CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NOSequences and Modification (5′–-3′) DN4 DN5 DN6 1 5′-TCG ₁AACG ₁TTCG₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 58.3 51.8 39.1 2 5′-TCG ₁AACG ₁TTCG-X-GCTTG₁CAAG ₁CT-5′ 62.5 56.5 43.6 3 5′-TCTCACCTTCT-X-TCTTCCACTCT-5′ 0 31.116.9 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 5 5′-TCG ₂AACG₂TTCG-X-GCTTG ₂CAAG ₂CT-5′ media 5.2 18.9 8.9 Normal phase represents aphosphorothioate linkage G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine X = Glycerol linker

TABLE 4B Immunomer Structure and Expression of BC from Human PBMC (2× 10⁶cell/ml) (24 hs) %CD86 %CD86 %CD86 SEQ Sequences and Modification 1μg/ml 1 μg/ml 1 μg/ml ID NO (5′–3′) DN1 DN2 DN3 6 5′-TCG ₁TCG ₁AACG₁TTCG ₁AGATGAT-3′ 43.4 25 34.6 7 5′-TCG ₂TCG ₂AACG ₂TTCG ₂AGATGAT-3′46.7 42.6 47.6 8 5′-TCG ₃TC%AACG ₃TTCG ₃AGATGAT-3′ 41.1 25.7 38.5 95′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′ 25 20.8 27.6 10 5′-TC ₂GTC ₂GAAC₂GTTC ₂GAGATGAT-3′ 36.4 22.2 26 11 5′-TC ₃GTC ₃GAAC ₃GTTC ₃GAGATGAT-3′30 17 22.2 media 10.7 8 7.6 %CD86 %CD86 %CD86 SEQ Sequences andModification 1 μg/ml 1 μg/ml 1 μg/ml ID NO (5′–3′) DN4 DN5 DN6 6 5′-TCG₁TCG ₁AACG ₁TTCG ₁AGATGAT-3′ 40 43.5 24.8 7 5′-TCG ₂TCG ₂AACG ₂TTCG₂AGATGAT-3′ 36.4 41 36.2 8 5′-TCG ₃TCG ₃AACG ₃TTCG ₃AGATGAT-3′ 57.1 30.434.9 9 5′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′ 13.3 12.1 15.2 10 5′-TC ₂GTC₂GAAC ₂GTTC ₂GAGATGAT-3′25 14.4 16.3 11 5′-TC ₃GTC ₃GAAC ₃GTTC₃GAGATGAT-3′ 18.1 15 16.8 media 52 3.9 6.8 %CD69 %CD69 %CD69 SEQSequences and Modification 1 μg/ml 1 μg/ml 1 μg/ml ID NO (5′–3′) DN1 DN2DN3 6 5′-TCG ₁TCG ₁AACG ₁TTCG ₁AGATGAT-3′ 56.4 43.8 68.7 7 5′-TCG ₂TCG₂AACG ₂TTCG ₂AGATGAT-3 55.6 58.6 65.5 8 5′-TCG ₃TCG ₃AACG ₃TTCG₃AGATGAT-3′ 50 39.3 73.1 9 5′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′ 15.4 2720 10 5′-TC ₂GTC ₂GAAC ₂GTTC ₂GAGATGAT-3′ 20 31.4 31.5 11 5′-TC ₃GTC₃GAAC ₃GTTC ₃GAGATGAT-3′ 10 22.2 24.3 media 0 11.1 11.1 %CD69 %CD69%CD69 SEQ Sequences and Modification 1 μg/ml 1 μg/ml 1 μg/ml ID NO(5′–3′) DN4 DN5 DN6 6 5′-TCG ₁TCG ₁AACG ₁TTCG ₁AGATGAT-3′ 57.1 7 5′-TCG₂TCG ₂AACG ₂TTCG ₂AGATGAT-3′ 60 8 5′-TCG ₃TCG ₃AACG ₃TTC%AGATGAT-3′ 37.59 5′-TC ₁GTC ₁GAAC ₁GTTC ₁GAGATGAT-3′ 15.4 10 5′-TC ₂GTC ₂GAAC ₂GTTC₂GAGATGAT-3′ 11.1 11 5′-TC ₃GTC ₃GAAC ₃GTTC ₃GAGATGAT-3′ 14.3 media 5.2Normal phase represents a phosphorothioate linkage G ₁= 2′-deoxy-7-deazaguanosine G ₂ = Arabinoguanosine G ₃ = 2′-deoxyinosineC ₁ = 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine C ₂= Arabinocytidine C ₃ = 2′-deoxy-5-hydroxycytidine

TABLE 4C Immunomer Structure and Expression of BC from Human PBN4C (2× 10⁶ cell/ml) (24 hs) %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml IDNO Sequences and Modification (5′–3′) DN1 DN2 DN3 1 5′-TCG ₁AACG ₁TTCG₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 32.3 34.8 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG₁CT-5′ 41.4 51.6 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 33.3 51.5 135′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 20 25.6 145′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 31.1 26.1 15 5′-TCG ₁TTG ₁CAACG ₁-X-G₁CAACG ₁TTG ₁CT-5′ 17.1 23.9 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG₂CT-5′ 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′media 19.4 20.9 %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NOSequences and Modification (5′–3′) DN4 DN5 DN6 1 5′-TCG ₁AACG ₁TTCG₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 15.4 33.3 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG₁CT-5′ 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG ₁AACG₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG₂CAAG ₂CT-5′ 30.7 45.4 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 3041.6 17 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ media 86 2.7 %CD86 %CD86 %CD86 SEQ 1μg/ml 1 μg/ml 1 μg/ml ID NO Sequences and Modification (5′–3′) DN7 DN8DN9 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 35.5 23.5 17.6 405′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 12 5′-TCG ₁AACG ₁TTC-X-CTTG₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 145′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 56.5 16 5′-TCG₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 46.7 17 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ media 9 2015.3 %CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences andModification (5′–3′) DN1 DN2 DN3 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG₁CT-5′ 23.5 64 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 20.8 62.5 125′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13.6 59 13 5′-TCG ₁TTCG ₁AACG₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 12.5 46.4 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′15.9 52.9 15 5′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 12.2 51.6 45′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 16 5′-TCG ₂AACG₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ media 14.8 34%CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences andModification (5′–3′) DN4 DN5 DN6 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG₁CT-5′ 53.8 62.5 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 12 5′-TCG₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG₁CT-5′ 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG ₁TTG ₁CAACG ₁-X-G₁CAACG ₁TTG ₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 77.770.6 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 57.1 64.7 17 5′-TCG₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC₁AAG ₁CT-5′ media 26.3 15 %CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/mlID NO Sequences and Modification (5′–3′) DN7 DN8 DN9 1 5′-TCG ₁AACG₁TTCG ₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 28.6 50 25 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG₁CAAG ₁CT-5′ 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG₁AACG ₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 155′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG ₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G₂CTTG ₂CAAG ₂CT-5′ 70.6 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 72.717 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG₁-X-G ₁CTTGC ₁AAG ₁CT-5′ media 14.1 13.2 12 Normal phase represents aphosphorothioate linkage G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= GlyceroI Linker

TABLE 4D Immunomer Structure and Expression of BC from Human PBMC (2× 10⁶ cell/ml) (24 hs) %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml IDNO Sequences and Modification (5′–3′) DN1 DN2 DN3 1 5′-TCG ₁AACG ₁TTCG₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 64.3 572 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG₁CT-5′ 59.2 58.3 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 49.3 40.9 135′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 25.3 24.7 145′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15.4 17.2 15 5′-TCG ₁TTG ₁CAACG ₁-X-G₁CAACG ₁TTG ₁CT-5′ 30.6 23.7 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG₂CT-5′ 16 5′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′media 2.6 13.9 %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NOSequences and Modification (5′–3′) DN4 DN5 DN6 1 5′-TCG ₁AACG ₁TTCG₁-X-G ₁CTTG ₁CAAG ₁CT-5′ 35.9 30.3 35.6 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG₁CAAG ₁CT-5′ 57.9 12 5′-TCG ₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 34.9 135′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG ₁CT-5′ 145′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG ₁TTG ₁CAACG ₁-X-G ₁CAACG ₁TTG₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 16 5-TCG ₂AACG₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5 media 12.3 11.1 14%CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences andModification (5′–3′) DN7 DN8 DN9 1 5′-TCG ₁AACG ₁TTCG ₁-X-G ₁CTTG ₁CAAG₁CT-5′ 28 32.3 40 5′-TCG ₁AACG ₁TTCT-X-TCTTG ₁CAAG ₁CT-5′ 12 5′-TCG₁AACG ₁TTC-X-CTTG ₁CAAG ₁CT-5′ 13 5′-TCG ₁TTCG ₁AACG ₁-X-G ₁CAAG ₁CTTG₁CT-5′ 14 5′-TCCAACCTTCG-X-GCTTCCAACCT-5′ 15 5′-TCG ₁TTG ₁CAACG ₁-X-G₁CAACG ₁TTG ₁CT-5′ 4 5′-TCG ₂AACG ₂TTCG ₂-X-G ₂CTTG ₂CAAG ₂CT-5′ 165′-TCG ₂AACG ₂TTCT-X-TCTTG ₂CAAG ₂CT-5′ 17 5′-TCG ₁AACG ₂TTCG ₁-X-G₁CTTG ₂CAAG ₁CT-5′ 18 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ media10.9 12.6 Normal phase represents a phosphorothioate linkage G ₁= 2′-deoxy-7-deazaguanosine G ₂ = Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= GlyceroI Linker

TABLE 4E Immunomer Structure and Expression of BC from Human PBMC assay(24 hs) %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequencesand Modification (5′–3′) DN1 DN2 DN3 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG₂CAAG ₁CT-5′ 20 9 34.6 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′21.7 12.5 31.4 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ and 34 225′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ and 35 23 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTGC ₁TGTCT-5′ and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ and37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG₁AACG ₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media 8.7 %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequencesand Modification (5′–3′) DN4 DN5 DN6 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG₂CAAG ₁CT-5′ 42.3 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 72.7 215′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 14.5 17.1 and 34 22 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 27.8 28.6 and 35 23 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTGC ₁TGTCT-5′ 28.9 22.2 and 36 24 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTCCACTCT-5′ 23.2 21.8 and 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 275′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 305′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 5.9 4.0 6.0 %CD86 %CD86%CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences and Modification(5′–3′) DN7 DN8 DN9 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 205′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₁CTGTCT-5′ 65 46.3 40.3 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG₂CTGTCT-5′ 75 49.2 46.5 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC₁TGTCT-5′ 78.9 54.3 45 and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′83.3 33.8 29.4 amd 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 3426 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 22.535.6 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 43.1 47.8 30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 325′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 4.6 0 10.5 %CD86 %CD86 %CD86 SEQ 1μg/ml 1 μg/ml 1 μg/ml ID NO Sequences and Modification (5′–3′) DN10 DN11DN12 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG₁CTGTCT-5′ 24.4 60.7 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′23.9 61.5 53.8 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 32.8 72and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 22.7 52.6 and 37 255′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ 31.7 and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ 23.9 and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 21 57.1 29.6 295′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 34.7 63.1 43.5 30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 24.5 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG₁T-5′ 28.6 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ 44.2 and 38 335′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ 28.3 and 39 Media 19 8.6 18%CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences andModification (5′–3′) DN1 DN2 DN3 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG₁CT-5′ 13 22.2 19.2 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 5230.7 59.3 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ and 34 22 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC₁TGTCT-5′ and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ and 37 255′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTfCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media 3 %CD69 %CD69 %CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequencesand Modification (5′–3′) DN4 DN5 DN6 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG₂CAAG ₁CT-5′ 76 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 85.1 215′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 35 20 and 34 22 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 57.3 39.4 and 35 23 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTGC ₁TGTCT-5′ 60.4 54.2 and 36 24 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTCCACTCT-5′ 69 30.2 and 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 275′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 305′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 10 59 10.1 %CD69 %CD69%CD69 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequences and Modification(5′–3′) DN7 DN8 DN9 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 205′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₁CTGTCT-5′ 88.2 47.7 59.7 and 34 22 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₂CTGTCT-5 97 55 63.3 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC₁TGTCT-5′ 96.8 68.3 60.2 and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′91.9 40.3 41.9 and 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 3426 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 3651.2 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 51.6 66.7 30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 325′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG₁C-Y-GACACG ₁TGTCT-5′ and 39 Media 9 10.3 11.1 %CD69 %CD69 %CD69 SEQ 1μg/ml 1 μg/ml 1 μg/ml ID NO Sequences and Modification (5′–3′) DN10 DN11DN12 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG₁CTGTCT-5′ 24 61.7 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′29.2 71.4 58 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 357 60.5and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 32.2 62.9 and 37 255′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ 23.6 and 34 26 5′-TG,CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ 16.7 and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 22.1 50 42.5 295′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 31.5 70.5 54.4 30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 19.5 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG₁T-5′ 15.5 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ 40 and 38 33 5′-TG₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ 19.3 and 39 Media 13.4 13.4 12.9Normal phase represents a phosphorothioate linkage; o represents aphosphodiester linkage. G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker Y = C ₃Linker

TABLE 4F Immunomer Structure and Expression of DC from Human PBMC assay(24 hs) %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequencesand Modification (5′–3′) DN1 DN2 DN3 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG₂CAAG ₁CT-5′ 11.9 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 12.5 215′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ and 34 22 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₂CTGTCT-5′ and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ and 37 25 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-XoCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media 13.7 %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NO Sequencesand Modification (5′–3′) DN4 DN5 DN6 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 54.7 68 and 34 22 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₂CTGTCT-5′ 58.8 75.3 and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC₁TGTCT-5′ 60.3 73.4 and 36 24 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 51.861.1 and 37 25 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media 33.7 62.8 %CD86 %CD86 %CD86 SEQ 1 μg/ml 1 μg/ml 1 μg/ml ID NOSequences and Modification (5′–3′) DN7 DN8 DN9 19 5′-TCG ₁AACG ₂TTCG₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 45.4 88.7 78.3 and 34 225′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 54.9 89.3 79.1 and 35 23 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 55.3 88.6 79.9 and 36 24 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTCCACTCT-5′ 47 85.7 n/a and 37 25 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG ₁C-Y-TCTTCCACTCT-5′and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG ₁T-5′ 28 5′-CTGTCG₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 82.1 29 5′-CTGTCoG ₂TTCTC-X-CTCTTG₂oCTGTC-5′ 89 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG₁TGTCT-5′ and 39 Media 47.5 56.1 53.2 %CD86 %CD86 %CD86 SEQ 1 μg/ml 1μg/ml 1 μg/ml ID NO Sequences and Modification (5′–3′) DN10 DN11 DN12 195′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG ₁-X-G₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 72 86.3and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 74.4 88.1 81.8 and 3523 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 77.1 87.5 and 36 24 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 68.3 83.8 and 37 25 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTG ₁CTGTCT-5′ 60.4 and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ 37.4 and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 61.1 79.6 58.2 295′-CTGTCoG ₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 68.2 87.3 69.5 30 5′-TCG ₁TGTCG₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 60.3 31 5′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG₁T-5′ 44.7 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG ₁CTGTCT-5′ 65.8 and 38 335′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ 49.1 and 39 Media 69.6 58.335.8 Normal phase represents a phosphorothioate linkage; o represents aphosphodiester linkage. G ₁ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁= 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X= Glycerol Linker Y = C ₃Linker

Example 11 B Cell Proliferation Assay

A total of 1×10⁵ B cells/200 μl were stimulated with 0.3, 1.0, 3.0, or10.0 μg/mL concentrations of immunomer compounds of the invention for 16hr, then pulsed with 0.75 μCi of [³H]-thymidine and harvested 8 h later.The incorporation of radioactivity was measured using liquidscintillation counter. Table 5 shows an average ±SD of B cellproliferation at a final concentration of 1.0 μg/mL.

TABLE 5 Immunomer Structure and Immunostimulatory Activity in HumanB-Cell Proliferation Assay (24 hs) [³H]T [³H]T [³H]T (cpm) (cpm) (cpm)SEQ ID 1 μg/ml 1 μg/ml 1 μg/ml NO Sequences and Modification (5′–3′) DN4DN5 DN6 19 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC₁GTTCG ₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG₁CTGTCT-5′ 31127 ± 6800 17626 ± 2809 and 34 22 5′-TCG ₁AACG ₁TTCG₁-Y-TCTTG ₂CTGTCT-5′ 33368 ± 1364 17131 ± 1366 and 35 23 5′-TCG ₁AACG₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 30845 ± 2541 13826 ± 2331 and 36 24 5′-TCG₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 34077 ± 3636 8073 ± 583 and 37 25 5′-TG₁CAAG ₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5 and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media  646 ± 236  457 ± 121 [³H]T [³H]T [³H]T (cpm) (cpm) (cpm) SEQ ID 1μg/ml 1 μg/ml 1 μg/ml NO Sequences and Modification (5′–3′) DN7 DN8 DN919 5′-TCG ₁AACG ₂TTCG ₁-X-G ₁CTTG ₂CAAG ₁CT-5′ 20 5′-TCG ₁AAC ₁GTTCG₁-X-G ₁CTTGC ₁AAG ₁CT-5′ 21 5′-TCG ₁AACG,TTCG ₁-Y-TCTTG ₁CTGTCT-5′ 37731± 2901 and 34 22 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTG ₂CTGTCT-5′ 38405 ± 8056and 35 23 5′-TCG ₁AACG ₁TTCG ₁-Y-TCTTGC ₁TGTCT-5′ 34702 ± 6196 and 36 245′-TCG ₁AACG ₁TTCG ₁-Y-TCTTCCACTCT-5′ 23030 ± 1941 and 37 25 5′-TG ₁CAAG₁CTTG ₁C-Y-TCTTG ₁CTGTCT-5′ and 34 26 5′-TG ₁CAAG ₁CTTG₁C-Y-TCTTCCACTCT-5′ and 37 27 5′-TG ₁CAAG ₁CTTG ₁C-X-CG ₁TTCG ₁AACG₁T-5′ 28 5′-CTGTCG ₂TTCTCo-X-oCTCTTG ₂CTGTC-5′ 29 5′-CTGTCoG₂TTCTC-X-CTCTTG ₂oCTGTC-5′ 30 5′-TCG ₁TGTCG ₁TTT-X-TTTG ₁CTGTG ₁CT-5′ 315′-TG ₁CTGTG ₁CTTT-X-TTTCG ₁TGTCG ₁T-5′ 32 5′-TCG ₁AACG ₁TTCG ₁-Y-GACAG₁CTGTCT-5′ and 38 33 5′-TG ₁CAACG ₁CTTG ₁C-Y-GACACG ₁TGTCT-5′ and 39Media  658 ± 205 Normal phase represents a phosphorothioate linkage; orepresents a phosphodiester linkage. G ₂ = 2′-deoxy-7-deazaguanosine G ₂= Arabinoguanosine C ₁ = 1-(2′-deoxy-62-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine X = Glycerol Linker Y = C₃Linker

EQUIVALENTS

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. An immunostimulatory oligonucleotide having the structure5′-TCG₁AACG₁TTCG₁-Y-GACAG₁CTGTCT-5′; wherein Y is a C3-linker, and G₁ is2′-deoxy-7-deazaguanosine.
 2. A formulation comprising theoligonucleotide according to claim 1 and a physiologically acceptablecarrier.
 3. A method for generating an immune response in a vertebrate,the method comprising administering to the vertebrate animmunostimulatory oligonucleotide having the structure5′-TCG₁AACG₁TTCG₁-Y-GACAG₁CTGTCT-5′; wherein Y is a C3-linker, and G₁ is2′-deoxy-7-deazaguanosine.
 4. The method according to claim 3, whereinthe route of administration is selected from parenteral, oral,sublingual, transdermal, topical, intranasal, aerosol, intraocular,intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye dropand mouthwash.
 5. The oligonucleotide according to claim 1, furthercomprising an antibody, antisense oligonucleotide, protein, antigen,allergen, chemotherapeutic agent or adjuvant.
 6. The compositionaccording to claim 2, further comprising an antibody, antisenseoligonucleotide, protein, antigen, allergen, chemotherapeutic agent oradjuvant.
 7. The method according to claim 3, further comprisingadministering an antibody, antisense oligonucleotide, protein, antigen,allergen, chemotherapeutic agent or adjuvant.